Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix

ABSTRACT

Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

RELATED APPLICATION DATA

This application is a continuation of PCT application no.PCT/US2016/060279, designating the United States and filed Nov. 3, 2016;which claims the benefit U.S. Provisional Patent Application No.62/250,182 filed on Nov. 3, 2015 each of which is hereby incorporatedherein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under grant number P50HG005550 awarded by National Institutes of Health, RC2HL102815 awardedby National Institutes of Health, MH098977 awarded by NationalInstitutes of Health, GM080177 awarded by National Institutes of Healthand DGE1144152 awarded by National Science Foundation. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for volumetricimaging of a three-dimensional matrix of nucleic acids where the nucleicacids have been amplified, detected and sequenced within the matrix.

BACKGROUND OF THE INVENTION

Since many gene products such as RNA and proteins are enriched inregions where they function, their location provides an important clueto their function. This property has been used for in situ fluorescenthybridization, immunohistochemistry and tissue-specific reporter assaysin numerous areas of biological research. Most optical sequencingmethods either utilize a two-dimensional solid substrate ormicrowells/microchambers to immobilize sequencing templates during thesequencing process in order to maintain spatial invariability foroptical detection, and therefore enable reconstruction of a singlenucleic acid template sequence. In both cases, optical detection ofsignal occurs in at most two two-dimensional planes.

SUMMARY

Embodiments of the present invention are directed to volumetric imagingof nucleic acids within a three-dimensional matrix, such as within afixed biological specimen, and an apparatus for volumetric imaging ofnucleic acids within a three-dimensional matrix, such as within a fixedbiological specimen. Volumetric imaging detects fluorescence- oroptically-encoded signals in three dimensions. According to one aspect,the three-dimensional positioning of a molecule within the threedimensional matrix is determined. According to certain aspects, methodsare provided for imaging an arbitrary volume in a method of volumetricdetection, imaging, and reconstruction. Exemplary volumetric detectionmethods include both methods for volumetric imaging of optical sectionsthat utilize optical sectioning (e.g. two-dimensional acquisition ofimages throughout a volume), as well as those that do not utilizeoptical sectioning (e.g. digital holography and physical sectioning).

Aspects of the present disclosure may include in situ nucleic acidsequencing methods using a three-dimensional matrix for immobilizationof nucleic acid sequencing templates during sequencing, maintaining thespatial relationships in three dimensions between sequencing templates,enabling detection and reconstruction of both sequences andthree-dimensional positional information. Exemplary methods describedherein are also directed to reconstruct three-dimensional biologicalfeatures, such as proteins and cell membranes. Exemplary volumetricimaging approaches include those described herein and those known in theart which measure light signals, systematically or otherwise, inthree-dimensional space. Accordingly, aspects of the present disclosureinclude a method and apparatus for volumetric imaging of in situsequenced nucleic acids. Aspects of the present disclosure furtherinclude an automated method and apparatus for volumetric imaging of insitu sequenced nucleic acids including an in situ sequencing method orapparatus, a fluidics method or reagent delivery method or apparatus toregulate flow of reagents and deliver reagents and a volumetric imagingmethod or apparatus for imaging and/or detecting light signals from thein situ sequenced nucleic acids or other molecules or structures ofinterest. According to certain aspects, the methods and apparatusdescribed herein are not limited to nucleic acids. Any molecule orstructure within a three dimensional matrix that can be detected, forexample, using detectable moieties, such as fluorescent moieties andother detectable moieties known to those of skill in the art can be thesubject of the volumetric imaging methods described herein. Suchmolecules or structures can include DNA, RNA, proteins, biomolecules,cellular structures and the like.

Exemplary methods of making a three dimensional matrix of nucleic acidsequences, amplifying such nucleic acid sequences, sequencing suchnucleic acid sequences and imaging such nucleic acid sequences areprovided in PCT US2014/18580 hereby incorporated by reference in itsentirety. Such methods include making a three dimensional matrixincluding nucleic acids covalently bound into a matrix or into or to amatrix material. The nucleic acids may be co-polymerized with the matrixmaterial or cross-linked to the matrix material or both. According toone aspect, a plurality of nucleic acid sequences of certain length,such as DNA or RNA sequences are part of a three-dimensional copolymer.According to one aspect, nucleic acids such as DNA or RNA sequences ofgiven length are covalently attached to a matrix material to preservetheir spatial orientation in the x, y and z axes within the matrix. Itis to be understood that the three dimensional matrix may include amatrix material and that the term copolymer, matrix and matrix materialmay be used interchangeably. Useful methods also include immobilizingnaturally occurring nucleic acids within their native environment, suchas within a cell or within a tissue sample. The three dimensionalnucleic acid matrix can be generated in situ in a cell or tissue sampleto preserve the naturally occurring nucleic acid sequence diversity(such as DNA and RNA) and spatial orientation in cells, tissues or anyother complex biomaterial. According to this aspect, the location ofnucleic acids and their relative position is identified as a threedimensional structure, such as within subcellular compartments, withincells, within tissues, as three dimensional nucleic acid assemblies, asthree dimensional nucleic acid material, etc. The nucleic acids can beamplified and sequenced, if desired, in situ thereby providingpositional information of the nucleic acids within the cell or tissue.

According to a related aspect, nucleic acids of interest or othermolecules of interest, whether naturally occurring or synthetic, can bepresent within a three dimensional matrix material and covalentlyattached to the three dimensional matrix material such that the relativeposition of each nucleic acid is fixed, i.e. immobilized, within thethree dimensional matrix material. In this manner, a three-dimensionalmatrix of covalently bound nucleic acids of any desired sequence isprovided. Each nucleic acid has its own three dimensional coordinateswithin the matrix material and each nucleic acid represents information.According to one aspect, individual nucleic acids, such as DNA or RNAcan be amplified and sequenced in situ, i.e., within the matrix.

According to a further aspect, the nucleic acids can be amplified toproduce amplicons within the three dimensional matrix material. Theamplicons can then be covalently attached to the matrix, for example, bycopolymerization or cross-linking. This results in a structurally stableand chemically stable three dimensional matrix of nucleic acids.According to this aspect, the three dimensional matrix of nucleic acidsallows for prolonged information storage and read-out cycles. Thenucleic acid/amplicon matrix allows for high throughput sequencing of awide ranging array of biological and non-biological samples in threedimensions.

According to certain aspects, a three dimensional nucleic acid matrix isprovided where a plurality of nucleic acid molecules, such as DNA orRNA, amplicons or nucleic acid structural units are immobilized, such asby covalent bonding to the matrix, in a three dimensional space relativeto one another. In this context, the nucleic acid molecules are rigidlyfixed to the extent that they maintain their coordinate position withinthe matrix. It is to be understood that even though a nucleic acidmolecule may be covalently attached to the three dimensional matrixmaterial, the nucleic acid molecule itself may be capable of movementthough bound to the matrix, such as for example, when a nucleic acidsequence is bound to the matrix at a single location on the nucleicacid.

According to one aspect, the three dimensional matrix including nucleicacids is porous. According to one aspect, the three dimensional matrixincluding nucleic acids is porous to the extent that reagents typicallyused in amplification methods can diffuse or otherwise move through thematrix to contact nucleic acids and thereby amplify nucleic acids undersuitable conditions. Porosity can result from polymerization and/orcrosslinking of molecules used to make the matrix material. Thediffusion property within the gel matrix is largely a function of thepore size. The molecular sieve size is chosen to allow for rapiddiffusion of enzymes, oligonucleotides, formamide and other buffers usedfor amplification and sequencing (>50-nm). The molecular sieve size isalso chosen so that large DNA or RNA amplicons do not readily diffusewithin the matrix (<500-nm). The porosity is controlled by changing thecross-linking density, the chain lengths and the percentage ofco-polymerized branching monomers according to methods known to those ofskill in the art.

According to one aspect, the three dimensional matrix material ischemically inert and thermally stable to allow for various reactionconditions and reaction temperatures. According to this aspect, thethree dimensional matrix material is chemically inert and thermallystable to conditions used in amplification and sequencing methods knownto those of skill in the art.

According to one aspect, the three dimensional matrix material isoptically transparent. According to one aspect, the three dimensionalmatrix material is optically transparent to allow for three dimensionalimaging techniques known to those of skill in the art.

According to one aspect, the nucleic acids are amplified to an extent toproduce sufficient levels of amplicons for three dimensional imaging.For example, the nucleic acids are amplified and include a labelsufficient for a high level of fluorescence compatible with threedimensional imaging.

According to one aspect, the material used to form the matrix iscompatible with a wide range of biological and non-biological specimensin situ so as to avoid extracting the nucleic acid molecules away fromtheir native environment.

According to one aspect, the matrix material may be a semi-solid mediumthat can be made from polyacrylamide, cellulose, alginate, polyamide,cross-linked agarose, cross-linked dextran or cross-linked polyethyleneglycol. In certain aspects, the semi-solid medium has x, y and z axes,and the nucleic acids are present randomly or non-randomly within thethree dimensional matrix.

In certain aspects, the semi-solid medium can be attached to a solidsupport such as a microscope slide or a flow cell. The solid support canbe attached to the bottom surface of the semi-solid medium.

According to one aspect, an automated method and device is provided forintroducing reagents into the matrix, such as for example, by usingfluidics or microfluidics devices including one or more reservoirs whichreagents are stored, channels or conduits to direct the reagents to thematrix and one or more pumps to force or draw the reagents from thereservoirs through the channels or conducts and to the matrix. Theautomated method and device may be controlled by a microprocessor andsoftware.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIG. 1 depicts in schematic the process of creating a matrix of nucleicacids within cells in situ, followed by amplifying the nucleic acids,such as DNA or RNA, in situ, co-polymerizing the amplicons in situ,covalently attaching the amplicons to the matrix material, interrogatingthe amplicons and imaging the amplicons along with a reconstructed 3Dcell image with DNA/RNA amplicons on the order of 10-7m.

FIG. 2 is a perspective view in schematic of components of an automatedsequencing and three-dimensional volumetric imaging device.

FIG. 3 is a front view in schematic of components of an automatedsequencing and three-dimensional volumetric imaging device.

FIG. 4 is a perspective view in schematic of components of an automatedsequencing and three-dimensional volumetric imaging device.

FIG. 5 is a perspective view in schematic of a stage with sample holdersof an automated sequencing and three-dimensional volumetric imagingdevice.

FIG. 6 is an exemplary schematic timing diagram useful with embodimentsdescribed herein.

FIG. 7 is an exemplary schematic block diagram illustrating aspects ofan exemplary TTL communication structure.

FIG. 8 depicts an exemplary timing and resource usage timing diagram forsequencing device fluidics, imaging, and dual stage subsystems.

FIG. 9 depicts an exemplary schematic block diagram illustrating thecontroller organization for the dual stages, fluidic, and imagingsubsystems.

FIG. 10 depicts exemplary device systems for managing the state of andinterfacing with an objective lens.

FIG. 11 depicts a flow chart for repeatable XYZ positioning of thesample relative to the imaging axis over time.

FIG. 12 depicts an exemplary system for mapping a surface of a sampleholder comprising a solid substrate.

DETAILED DESCRIPTION

The present invention provides a method and apparatus for analysis ofmolecules present within a three-dimensional matrix, such as a pluralityof nucleic acids within a three-dimensional matrix. According to oneaspect, an automated sequencing and three-dimensional imaging device(i.e., volumetric imaging device) is provided that can measure, resolveand optionally localizing light signals in three-dimensional space tomeasure fluorescently- or optically-encoded nucleic-acid sequencingwithin a three-dimensional matrix.

According to one aspect, the nucleic acids have been amplified andsequenced using optical sequencing methods known to those of skill inthe art so that the nucleic acid can be optically detected. According toone aspect, an automated sequencing and three-dimensional imaging deviceis provided which includes a three-dimensional imaging apparatus,apparatus for fluid exchange, apparatus for temperature control, andapparatus for computational analysis. The automated sequencing andthree-dimensional imaging device uses biochemical methods to executefluorescence- or optically-encoded nucleic acid sequencing, acquisitionof image data, and optionally, data processing.

According to one aspect, a plurality of nucleic acids within athree-dimensional matrix that have been amplified and sequenced usingoptical sequencing methods known to those of skill in the art so thatthe nucleic acid can be optically detected is volumetrically imaged.According to this aspect, during detection of fluorescence- oroptically-encoded nucleic acid sequencing chemistry, light emanates froma nucleic acid template molecule or multimolecular amplicon in threedimensions upon excitation. The three-dimensional distribution ofemission light, called a point spread function, is created by an opticalsystem when imaging a point source. In traditional wide-field microscopysystems, as the focal plane distance increases from the point source inZ, the image becomes less point-like. The total integrated intensity ateach out-of-focus plane is the same as that at the focal plane, but inpractice the intensity level drops below the sensitivity of the detectorafter a certain distance. The out-of-focus light is also dispersed,enabling resolution of objects in another focal plane whose intensity isgreater than the out-of-focus background. The size and shape of thepoint spread function is determined by the optical system, especiallythe numerical aperture of the objective. Although wide-field microscopycan be used to image a volume in this way, additional methods achievegreater axial resolution and signal-to-noise.

Exemplary volumetric imaging approaches used in the automated sequencingand three-dimensional imaging device described herein are broadlydivided into two categories: structured illumination and emissionmanipulation. Additional approaches include computational reconstructionand measurement of angular components of emission light. Theseapproaches provide a systematic measurement of the intensity andwavelength of light in three-dimensional space.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using 3DStructured Illumination (3DSIM). In 3DSIM, spatially patterned light isused for excitation, and fringes in the Moiré pattern generated byinterference of the illumination pattern and the sample, are used toreconstruct the source of light in three dimensions. Multiple spatialpatterns are used to excite the same physical region in order toilluminate the whole field. Digital processing or analog methods areused to reconstruct the final image. See York, Andrew G., et al.“Instant super-resolution imaging in live cells and embryos via analogimage processing.” Nature methods 10.11 (2013): 1122-1126 andGustafsson, Mats G L, et al. “Three-dimensional resolution doubling inwide-field fluorescence microscopy by structured illumination.”Biophysical journal 94.12 (2008): 4957-4970 each of which are herebyincorporated by reference in their entireties.

Two-photon, or multi-photon, microscopic modalities are usefulstructured illumination microscopy methods. See Denk W., Strickler J.,Webb W. (1990). “Two-photon laser scanning fluorescence microscopy”.Science 248 (4951): 73-6 hereby incorporated by reference in itsentirety. Two-photon microscopy is a type of microscopy that enablesimaging deep within a sample by using two photons per excitation event.These systems typically use long-wavelength light for excitation, whichpenetrates more effectively into tissue due to reduced scattering. Theuse of two-photon excitation also reduces background signal assingle-photon absorption provides insufficient energy to excite emissionby the fluorophore. Two-photon microscopy can also utilize larger ormore efficient optical and sensor configurations to detect the emission,as the localization of excitation over time is known to the imagingsystem during scanning. Other benefits to this modality include reducedphotodamage to the sample.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using a planarillumination method such as selective planar illumination microscopy(SPIM) or light sheet microscopy (LSM). In SPIM, optical detectingmoieties are excited selectively in each plane in a third dimensionwhile a two-dimensional image is acquired in the plane orthogonal to theillumination axis, providing effective separation over imagingtime/frames between objects distributed in the third dimension. SeeHuisken, Jan, et al. “Optical sectioning deep inside live embryos byselective plane illumination microscopy.” Science 305.5686 (2004):1007-1009 hereby incorporated by reference in its entirety. Systematicsequential imaging of planes provides for volumetric measurement. Theplanar illumination light may be generated using any number ofapproaches such as Gaussian and Bessel beam shaping.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using emissionmanipulation, such as confocal microscopy where one or more pinholes arepositioned at the confocal plane of the lens, blocking out-of-focuslight from reaching the detector. The focal plane of the lens issystematically shifted through the third dimension, enabling volumetricimaging. See Wilson, Tony. “Confocal microscopy.” Academic Press:London, etc 426 (1990): 1-64 hereby incorporated by reference in itsentirety.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a confocal imaging ormicroscopy modality. According to a certain aspect, the confocal imagingor microscopy modality is a scanning laser confocal modality. Accordingto another aspect, the confocal modality is a spinning disk confocalmodality. The spinning disk may be a Nipkow disk. According to anotheraspect, the confocal microscopy modality is a parallel beam scanninglaser modality, wherein two or more pinholes are scanned across thesample, such as by using a mirror galvanometer. The confocal modalitymay comprise one or more microlens arrays, such as for focusingexcitation light onto the pinhole array.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using a parallelconfocal method including aperture correlation. See Wilson, Tony, et al.“Confocal microscopy by aperture correlation.” Optics letters 21.23(1996): 1879-1881 hereby incorporated by reference in its entirety.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using a microlensarray for volumetric imaging, known as light field microscopy. See,Broxton et al. (2013) “Wave Optics Theory and 3-D Deconvolution for theLight Field Microscope” Stanford Computer Graphics Laboratory TechnicalReport 2013-1 hereby incorporated by reference in its entirety.According to this aspect, a microlens array between the main lens andthe detector pass light, which would otherwise focus at an intermediateplane, onto the light field for detection. 3D reconstruction algorithmsare applied to generate a volumetric image.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using a method ofvolumetric reconstruction from slices. According to this aspect ofimaging a volume of arbitrary dimension, a specimen may be sectionedinto segments of arbitrary dimension for the purpose of imaging. Theoriginal volume is reconstructed using information about the relativeposition of each segment imaged with respect to the original volume.This process is typically referred to as “acquiring serial sections,”and is used due to limitations in depth of scanning of volumetricimaging modalities, as well as by limitations of manipulating andlabeling thick samples, where diffusion may limit penetration ofreagents deep into specimens. In extraordinary cases, the sections maybe thinner than the diffraction limit of light, enabling resolution inthe axis of sectioning beyond what is achievable by the diffractionlimit of light. According to the 3D volumetric reconstruction fromserial sections method described herein, the sample is sectioned eitherbefore or after creation of the 3D sequencing library, but specificallyin such a way that the spatial relationship between sections ispreserved. For example, each section is placed in a separate well of aflowcell and given a unique identification. During sectioning, thesamples are attached onto a solid support substrate, particularly bycreation of covalent cross-links between the sample matrix and the solidsubstrate or by creation of a new encapsulating structural matrix.According to one aspect, the section is transferred onto functionalizedglass and covalent chemical cross-links are formed between the glass andthe 3D matrix of the sample. According to one aspect, the section istransferred onto functionalized glass and a new supporting matrix, whichis covalently linked to the glass surface, is formed to encapsulate thesample and provide structural support. For example, a 4% 1:19acrylamide:bis gel is formed around and through a section of abiological sample as the primary 3D matrix for FISSEQ; or a pre-existingFISSEQ gel (including those formed using a polyacrylamide matrix as theprimary 3D matrix) is sectioned and encased in a secondary 3Dstabilizing matrix.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged usingdeconvolution microscopy including computational algorithmic methods ofprocessing digital image data so as to remove the effect of the opticalcomponents of the microscope. See Biggs, David S C. “3D deconvolutionmicroscopy.” Current Protocols in Cytometry (2010): 12-19 herebyincorporated by reference in its entirety. Because optical microscopydetects point sources of light as a point spread function that exists inthree dimensions, deconvolution methods described herein reassign out offocus light to the point source, often using models or measurements ofthe point spread function of the optical system. This can serve toeffectively increase resolution in three dimensions.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged usingaberration-corrected multifocus microscopy to simultaneously captureimages from multiple sample planes, using Diffractive Fourier optics tocreate an instant array of focal of 2D wide-field images, recordedsimultaneously in one or more camera frames. See Abrahamsson, Sara, etal. “Fast multicolor 3D imaging using aberration-corrected multifocusmicroscopy.” Nature methods 10.1 (2013): 60-63 hereby incorporated byreference in its entirety. As with microlens array microscopy, theentire imaging volume is recorded without mechanical movement of parts.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which includes a three-dimensional imagingapparatus where the three-dimensional matrix is imaged using digitalholographic microscopy. According to this aspect, digital holographicmicroscopy does not record a projected image, but rather records thelight wave front information as a hologram. See Manoharan “DigitalHolographic Microscopy for 3D Imaging of Complex Fluids and BiologicalSystems” hereby incorporated by reference in its entirety. The amplitudeand phase of light is measured using digital sensors. The hologramcontains all information needed for reconstruction of the volume. If theobject wave front is measured from multiple angles, all opticalcharacteristics of the object may be fully characterized. Because thereis no image-forming lens, reconstruction algorithms that model theoptical system will reconstruct the volume without aberration.

According to one aspect, the automated sequencing and three-dimensionalimaging device described herein can carry out one or more volumetricimaging methods, such as by including one or more volumetric imagingapparatuses to carry out one or more volumetric imaging methods. Acombination of volumetric imaging methods as described herein may beused to further enhance resolution, imaging speed, light efficiency, orgain additional benefits. For example, SIM and confocal microscopyprinciples have been combined into multifocal SIM (mSIM). See York,Andrew G., et al. “Resolution doubling in live, multicellular organismsvia multifocal structured illumination microscopy.” Nature methods 9.7(2012): 749-754 hereby incorporated by reference in its entirety. Also,volumetric reconstruction of slices may be combined with other methods,such as confocal microscopy, since confocal microscopy has a depth limitof hundreds of microns, while it is desirable to sequence withinthree-dimensional matrices of arbitrary dimensions.

According to one aspect, the automated sequencing and three-dimensionalimaging device described herein comprises an imaging modality with oneimage sensor. According to another aspect, the automated sequencing andthree-dimensional imaging device described herein comprises an imagingmodality with two or more image sensors. According to one aspect, theautomated sequencing and three-dimensional imaging device describedherein comprises an imaging modality with four image sensors. Accordingto one aspect, the image sensor is a photon multiplier tube (PMT).According to another aspect, the image sensor is a charge-coupled device(CCD). According to a separate aspect, the image sensor is aComplementary metal-oxide-semiconductor (CMOS). According to one aspect,the image sensor is cooled by an integrated air or liquid coolingapparatus, such as for the purpose of reducing electrical noise or tostabilize the thermal operating conditions of the sensor. Furtheraccording to this aspect, a liquid cooling apparatus may provide forcooling with reduced vibration relative to active air cooling, such asby a fan. The cooling apparatus or unit may use a heat sink inconjunction with a fan to dissipate heat produced during temperaturechanges. The heating or cooling apparatus or unit may use a radiator andliquid cooling/circulating system to dissipate heat produced duringtemperature changes. The heating or cooling apparatus or unit may usetemperature sensors or thermistors to provide temperature feedback to acontrol system, which may be a microcontroller or built-to-taskelectronic circuit.

According to one aspect, the automated sequencing and three-dimensionalimaging device described herein comprises a color multiplexer fordetection of one or more distinct colors of light. The light emittedfrom one or more fluorescent emitters is detected by a detector. Incertain aspects, the detector is configured to detect photons of lightwith certain wavelengths. According to another aspect, the emissionlight is filtered such that only photons of certain wavelengths aredetected by a photon detector. Further according to another aspect, thedevice contains one or more emission filters, excitation filters, and/ordichroics for directing certain wavelengths of light within the opticalsystem. Further according to one aspect, the device contains one or moreacousto-optical tunable filter(s) (AOTF) for directing certainwavelengths of light to a certain detector or detectors. According toone aspect, colors of light signals are detected in serial. According toanother aspect, two or more distinguishable colors of light are detectedin parallel by one or more sensors.

According to one aspect, the automated sequencing and three-dimensionalimaging device described herein is used to detect two or more colors oflight in serial or parallel. According to one aspect, the colors oflight being detected are spaced along the electromagnetic spectrum tofacilitate discrimination between the colors. According to certainaspects, the fluorescence signals are spaced along the electromagneticspectrum by emission wavelength to facilitate specific detection ofcertain fluorescent moieties. According to certain aspects, thefluorescence signals are spaced along the electromagnetic spectrum byexcitation wavelength to facilitate specific excitation of certainfluorescent moieties. According to one exemplary aspect, the colors ofemission light are distributed around about 510 nm, 570 nm, 620 nm,and/or 680 nm. According to another exemplary aspect, the colors ofexcitation light are distributed around about 480 nm, 530 nm, 590 nm,and/or 640 nm.

According to one aspect, the automated sequencing and three-dimensionalimaging device described herein comprises one or more sources of light.According to one aspect, the light source is used for the purpose ofexciting fluorescence emission by the sample. According to anotheraspect, the light source is used for the purpose of detectingabsorbance, Raman scattering, or other modalities of interaction betweenthe light and the sample. According to a certain aspect, the lightsource is comprised of one or more light emitting diodes (LED).

According to another aspect, the light source is comprised of one ormore lasers. According to another aspect, the light source is comprisedof one or more lamps, such as a mercury or metal halide lamp. Accordingto a certain aspect, the light source is coupled to the device by freespace optics, wherein the light is propagated through gas or vacuum fromthe source to the optical system of the device. According to anotheraspect, the light source is coupled to the device by a fiber optic orliquid light guide. According to an exemplary aspect, the light istransmitted in its entirety or in part along a fiber optic with a squarecore. Further according to this aspect, the square core is paired with asquare aperture or a square field stop. Further according to thisaspect, the dimensions of the square core and/or square aperture may bedesigned to match the dimensions of the image sensor within the opticalsystem. According to certain aspects, multiple fibers are fused as amechanism of combining multiple colors of light into a single fiber.According to certain aspects, multiple fibers are optically combined asa mechanism of combining multiple colors of light into a single fiber.According to certain aspects, the excitation light is passed through ashutter to control the propagation of the light into or through theoptical system. According to one aspect, an acousto-optical tunablefilter (AOTF) is used to control the propagation of the light into orthrough the optical system. According to certain aspects, thepropagation of the excitation light into or through the optical systemis mechanically, electrically, or electromechanically coupled with acontroller system for the purpose of synchronizing one or more eventswithin the device.

According to one aspect, the sample to be analyzed by the automatedsequencing and three-dimensional imaging device described herein is athree dimensional matrix including a plurality of nucleic acids boundthereto. According to one aspect, the matrix is a three dimensionalnucleic acid-containing polymer. The nucleic acids may be naturallyoccurring nucleic acids or non-naturally occurring nucleic acids, suchas nucleic acids that have been made using synthetic methods. Thenucleic acids in the three dimensional matrix may be ordered orunordered. The nucleic acids in the three dimensional matrix may bepresent in their natural spatial relationship within a cell, tissue ororganism. The nucleic acids in the three dimensional matrix may bepresent in rows and columns within the three dimensional matrix such aswith a regular or repeating array.

According to one aspect, the nucleic acids are modified to incorporate afunctional moiety for attachment to the matrix. The functional moietycan be covalently cross-linked, copolymerize with or otherwisenon-covalently bound to the matrix. The functional moiety can react witha cross-linker. The functional moiety can be part of a ligand-ligandbinding pair. dNTP or dUTP can be modified with the functional group, sothat the function moiety is introduced into the DNA duringamplification. A suitable exemplary functional moiety includes an amine,acrydite, alkyne, biotin, azide, and thiol. In the case of crosslinking,the functional moiety is cross-linked to modified dNTP or dUTP or both.Suitable exemplary cross-linker reactive groups include imidoester(DMP), succinimide ester (NHS), maleimide (Sulfo-SMCC), carbodiimide(DCC, EDC) and phenyl azide. Cross-linkers within the scope of thepresent disclosure may include a spacer moiety. Such spacer moieties maybe functionalized. Such spacer moieties may be chemically stable. Suchspacer moieties may be of sufficient length to allow amplification ofthe nucleic acid bound to the matrix. Suitable exemplary spacer moietiesinclude polyethylene glycol, carbon spacers, photo-cleavable spacers andother spacers known to those of skill in the art and the like.

According to one aspect, a matrix-forming material is contacted to aplurality of nucleic acids spatially arrange in three-dimensionsrelative to one another.

Matrix forming materials include polyacrylamide, cellulose, alginate,polyamide, cross-linked agarose, cross-linked dextran or cross-linkedpolyethylene glycol. The matrix forming materials can form a matrix bypolymerization and/or crosslinking of the matrix forming materials usingmethods specific for the matrix forming materials and methods, reagentsand conditions known to those of skill in the art.

According to one aspect, a matrix-forming material can be introducedinto a cell. The cells are fixed with formaldehyde and then immersed inethanol to disrupt the lipid membrane. The matrix forming reagents areadded to the sample and are allowed to permeate throughout the cell. Apolymerization inducing catalyst, UV or functional cross-linkers arethen added to allow the formation of a gel matrix. The un-incorporatedmaterial is washed out and any remaining functionally reactive group isquenched. Exemplary cells include any cell, human or otherwise,including diseased cells or healthy cells. Certain cells include humancells, non-human cells, human stem cells, mouse stem cells, primary celllines, immortalized cell lines, primary and immortalized fibroblasts,HeLa cells and neurons.

According to one aspect, a matrix-forming material can be used toencapsulate a biological sample, such as a tissue sample. Theformalin-fixed embedded tissues on glass slides are incubated withxylene and washed using ethanol to remove the embedding wax. They arethen treated with Proteinase K to permeabilized the tissue. Apolymerization inducing catalyst, UV or functional cross-linkers arethen added to allow the formation of a gel matrix. The un-incorporatedmaterial is washed out and any remaining functionally reactive group isquenched.

Exemplary tissue samples include any tissue samples of interest whetherhuman or non-human. Such tissue samples include those from skin tissue,muscle tissue, bone tissue, organ tissue and the like. Exemplary tissuesinclude human and mouse brain tissue sections, embryo sections, tissuearray sections, and whole insect and worm embryos.

The matrix-forming material forms a three dimensional matrix includingthe plurality of nucleic acids. According to one aspect, thematrix-forming material forms a three dimensional matrix including theplurality of nucleic acids while maintaining the spatial relationship ofthe nucleic acids. In this aspect, the plurality of nucleic acids isimmobilized within the matrix material. The plurality of nucleic acidsmay be immobilized within the matrix material by co-polymerization ofthe nucleic acids with the matrix-forming material. The plurality ofnucleic acids may also be immobilized within the matrix material bycrosslinking of the nucleic acids to the matrix material or otherwisecross-linking with the matrix-forming material. The plurality of nucleicacids may also be immobilized within the matrix by covalent attachmentor through ligand-protein interaction to the matrix.

According to one aspect, the matrix is porous thereby allowing theintroduction of reagents into the matrix at the site of a nucleic acidfor amplification of the nucleic acid. A porous matrix may be madeaccording to methods known to those of skill in the art. In one example,a polyacrylamide gel matrix is co-polymerized with acrydite-modifiedstreptavidin monomers and biotinylated DNA molecules, using a suitableacrylamide:bis-acrylamide ratio to control the cross-linking density.Additional control over the molecular sieve size and density is achievedby adding additional cross-linkers such as functionalized polyethyleneglycols. According to one aspect, the nucleic acids, which may representbits of information, are readily accessed by oligonucleotides, such aslabeled oligonucleotide probes, primers, enzymes and other reagents withrapid kinetics.

According to one aspect, the matrix is sufficiently opticallytransparent or otherwise has optical properties suitable for standardNext Generation sequencing chemistries and deep three dimensionalimaging for high throughput information readout. The Next Generationsequencing chemistries that utilize fluorescence imaging include ABISoLiD (Life Technologies), in which a sequencing primer on a template isligated to a library of fluorescently labeled oligonucleotides with acleavable terminator. After ligation, the beads are then imaged usingfour color channels (FITC, Cy3, Texas Red and Cy5). The terminator isthen cleaved off leaving a free-end to engage in the nextligation-extension cycle. After all dinucleotide combinations have beendetermined, the images are mapped to the color code space to determinethe specific base calls per template. The workflow is achieved using anautomated fluidics and imaging device (i.e. SoLiD 5500 W GenomeAnalyzer, ABI Life Technologies). Another sequencing platform usessequencing by synthesis, in which a pool of single nucleotide with acleavable terminator is incorporated using DNA polymerase. Afterimaging, the terminator is cleaved and the cycle is repeated. Thefluorescence images are then analyzed to call bases for each DNAamplicons within the flow cell (HiSeq, Illumia).

According to certain aspects, the plurality of nucleic acids may beamplified to produce amplicons by methods known to those of skill in theart. The amplicons may be immobilized within the matrix generally at thelocation of the nucleic acid being amplified, thereby creating alocalized colony of amplicons. The amplicons may be immobilized withinthe matrix by steric factors. The amplicons may also be immobilizedwithin the matrix by covalent or noncovalent bonding. In this manner,the amplicons may be considered to be attached to the matrix. By beingimmobilized to the matrix, such as by covalent bonding or crosslinking,the size and spatial relationship of the original amplicons ismaintained. By being immobilized to the matrix, such as by covalentbonding or crosslinking, the amplicons are resistant to movement orunraveling under mechanical stress.

According to one aspect, the amplicons, such as DNA amplicons, are thencopolymerized and/or covalently attached to the surrounding matrixthereby preserving their spatial relationship and any informationinherent thereto. For example, if the amplicons are those generated fromDNA or RNA within a cell embedded in the matrix, the amplicons can alsobe functionalized to form covalent attachment to the matrix preservingtheir spatial information within the cell thereby providing asubcellular localization distribution pattern.

As used herein, the term “attach” refers to both covalent interactionsand noncovalent interactions. A covalent interaction is a chemicallinkage between two atoms or radicals formed by the sharing of a pair ofelectrons (i.e., a single bond), two pairs of electrons (i.e., a doublebond) or three pairs of electrons (i.e., a triple bond). Covalentinteractions are also known in the art as electron pair interactions orelectron pair bonds. Noncovalent interactions include, but are notlimited to, van der Waals interactions, hydrogen bonds, weak chemicalbonds (i.e., via short-range noncovalent forces), hydrophobicinteractions, ionic bonds and the like. A review of noncovalentinteractions can be found in Alberts et al., in Molecular Biology of theCell, 3d edition, Garland Publishing, 1994, incorporated herein byreference in its entirety for all purposes.

As used herein, the term “nucleic acid” includes the term“oligonucleotide” or “polynucleotide” which includes a plurality ofnucleotides. The term “nucleic acid” is intended to include naturallyoccurring nucleic acids and synthetic nucleic acids. The term “nucleicacid” is intended to include single stranded nucleic acids and doublestranded nucleic acids. The term “nucleic acid” is intended to includeDNA and RNA, whether single stranded or double stranded. Nucleotides ofthe present invention will typically be the naturally-occurringnucleotides such as nucleotides derived from adenosine, guanosine,uridine, cytidine and thymidine. When oligonucleotides are referred toas “double-stranded,” it is understood by those of skill in the art thata pair of oligonucleotides exists in a hydrogen-bonded, helical arraytypically associated with, for example, DNA. In addition to the 100%complementary form of double-stranded oligonucleotides, the term“double-stranded” as used herein is also meant to include those formwhich include such structural features as bulges and loops (see Stryer,Biochemistry, Third Ed. (1988), incorporated herein by reference in itsentirety for all purposes). As used herein, the term “polynucleotide”refers to a strand of nucleic acids that can be a variety of differentsizes. Polynucleotides may be the same size as an oligonucleotide, ormay be two-times, three-times, four-times, five-times, ten-times, orgreater than the size of an oligonucleotide.

Oligonucleotides and/or polynucleotides may be isolated from naturalsources or purchased from commercial sources. Oligonucleotide and/orpolynucleotide sequences may be prepared by any suitable method, e.g.,the phosphoramidite method described by Beaucage and Carruthers ((1981)Tetrahedron Lett. 22: 1859) or the triester method according toMatteucci et al. (1981) J. Am. Chem. Soc. 103:3185), both incorporatedherein by reference in their entirety for all purposes, or by otherchemical methods using either a commercial automated oligonucleotidesynthesizer or high-throughput, high-density array methods describedherein and known in the art (see U.S. Pat. Nos. 5,602,244, 5,574,146,5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and4,659,774, incorporated herein by reference in its entirety for allpurposes). Pre-synthesized oligonucleotides may also be obtainedcommercially from a variety of vendors.

In certain embodiments of the invention oligonucleotides and/orpolynucleotides may be prepared using a variety of microarraytechnologies known in the art. Pre-synthesized oligonucleotide and/orpolynucleotide sequences may be attached to a support or synthesized insitu using light-directed methods, flow channel and spotting methods,inkjet methods, pin-based methods and bead-based methods set forth inthe following references: McGall et al. (1996) Proc. Natl. Acad. Sci.U.S.A. 93:13555; Synthetic DNA Arrays In Genetic Engineering, Vol.20:111, Plenum Press (1998); Duggan et al. (1999) Nat. Genet. S21:10;Microarrays: Making Them and Using Them In Microarray Bioinformatics,Cambridge University Press, 2003; U.S. Patent Application PublicationNos. 2003/0068633 and 2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890,6,824,866, 6,800,439, 6,375,903 and 5,700,637; and PCT Application Nos.WO 04/031399, WO 04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO03/065038, WO 03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO03/040410 and WO 02/24597; incorporated herein by reference in theirentirety for all purposes.

Nucleic acids may be obtained from libraries, e.g., genomic libraries,cDNA libraries and the like. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc.Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem.37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.37:1233, incorporated herein by reference in their entirety for allpurposes.

In certain embodiments, nucleic acids are those found naturally in abiological sample, such as a cell or tissue.

In still other aspects, a matrix is used in conjunction with a solidsupport. For example the matrix can be polymerized in such a way thatone surface of the matrix is attached to a solid support (e.g., a glasssurface), while the other surface of the matrix is exposed or sandwichedbetween two solid supports. According to one aspect, the matrix can becontained within a container.

Solid supports of the invention may be fashioned into a variety ofshapes. In certain embodiments, the solid support is substantiallyplanar. Examples of solid supports include plates such as slides,microtitre plates, flow cells, coverslips, microchips, and the like,containers such as microfuge tubes, test tubes and the like, tubing,sheets, pads, films and the like. Additionally, the solid supports maybe, for example, biological, nonbiological, organic, inorganic, or acombination thereof.

Embodiments of the present invention are further directed to theamplification of nucleic acid sequences within the matrix, i.e. in situ,within the matrix. Methods of amplifying nucleic acids include rollingcircle amplification in situ. In certain aspects, methods of amplifyingnucleic acids involves the use of PCR, such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364; incorporated herein byreference in their entirety for all purposes). Alternative amplificationmethods include: self-sustained sequence replication (Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87:1874, incorporated herein byreference in its entirety for all purposes), transcriptionalamplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. US.86:1173, incorporated herein by reference in its entirety for allpurposes), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197,incorporated herein by reference in its entirety for all purposes),recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williamset al. (2002) J. Biol. Chem. 277:7790; incorporated herein by referencein their entirety for all purposes) or any other nucleic acidamplification method using techniques well known to those of skill inthe art. A variety of amplification methods are described in U.S. Pat.Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and5,612,199, incorporated herein by reference in their entirety for allpurposes. Embodiments of the present invention are directed to methodsof amplifying nucleic acids in situ within the matrix by contacting thenucleic acids within the matrix with reagents, such as primers andnucleotides, and under suitable reaction conditions sufficient toamplify the nucleic acids. According to one aspect, the matrix is porousto allow migration of reagents into the matrix to contact the nucleicacids.

In accordance with certain examples, methods of sequencing a nucleicacid in situ within a matrix are provided. General sequencing methodsknown in the art, such as sequencing by extension with reversibleterminators, fluorescent in situ sequencing (FISSEQ), pyrosequencing,massively parallel signature sequencing (MPSS) and the like (describedin Shendure et al. (2004) Nat. Rev. 5:335, incorporated herein byreference in its entirety), are suitable for use with the matrix inwhich the nucleic acids are present. Reversible termination methods usestep-wise sequencing-by-synthesis biochemistry that coupled withreversible termination and removable fluorescence (Shendure et al. supraand U.S. Pat. Nos. 5,750,341 and 6,306,597, incorporated herein byreference. FISSEQ is a method whereby DNA is extended by adding a singletype of fluorescently-labelled nucleotide triphosphate to the reaction,washing away unincorporated nucleotide, detecting incorporation of thenucleotide by measuring fluorescence, and repeating the cycle. At eachcycle, the fluorescence from previous cycles is bleached or digitallysubtracted or the fluorophore is cleaved from the nucleotide and washedaway. FISSEQ is described further in Mitra et al. (2003) Anal. Biochem.320:55, incorporated herein by reference in its entirety for allpurposes. Pyrosequencing is a method in which the pyrophosphate (PPi)released during each nucleotide incorporation event (i.e., when anucleotide is added to a growing polynucleotide sequence). The PPireleased in the DNA polymerase-catalyzed reaction is detected by ATPsulfurylase and luciferase in a coupled reaction which can be visiblydetected. The added nucleotides are continuously degraded by anucleotide-degrading enzyme. After the first added nucleotide has beendegraded, the next nucleotide can be added. As this procedure isrepeated, longer stretches of the template sequence are deduced.Pyrosequencing is described further in Ronaghi et al. (1998) Science281:363, incorporated herein by reference in its entirety for allpurposes. MPSS utilizes ligation-based DNA sequencing simultaneously onmicrobeads. A mixture of labelled adaptors comprising all possibleoverhangs is annealed to a target sequence of four nucleotides. Thelabel is detected upon successful ligation of an adaptor. A restrictionenzyme is then used to cleave the DNA template to expose the next fourbases. MPSS is described further in Brenner et al. (2000) Nat. Biotech.18:630, incorporated herein by reference in its entirety for allpurposes.

According to certain aspects, the nucleic acids within the matrix can beinterrogated using methods known to those of skill in the art includingfluorescently labeled oligonucleotide/DNA/RNA hybridization, primerextension with labeled ddNTP, sequencing by ligation and sequencing bysynthesis. Ligated circular padlock probes described in Larsson, et al.,(2004), Nat. Methods 1:227-232 can be used to detect multiple sequencetargets in parallel, followed by either sequencing-by-ligation,-synthesis or -hybridization of the barcode sequences in the padlockprobe to identify individual targets.

FIG. 1 depicts in schematic the process of creating a matrix of nucleicacids within cells in situ, followed by amplifying the nucleic acids,such as DNA or RNA, in situ, co-polymerizing the amplicons in situ,covalently attaching the amplicons to the matrix material, interrogatingthe amplicons and imaging the amplicons along with a reconstructed 3Dcell image with DNA/RNA amplicons on the order of 10-7m. According tocertain aspects, FISSEQ methods and materials useful in the practice ofthe methods described herein are provided in Lee et al., NatureProtocols, vol. 10, No. 3 (2015) pp. 442-458, Lee et al., Science 343,1360-1363 (2014) and Supplementary Materials published 27 Feb. 2014 onScience Express DOI: 10.1126/scienmce.1250212 each of which are herebyincorporated by reference in its entirety.

The automated sequencing and three-dimensional imaging device may beused for detecting optical signals distributed in three dimensions. Theautomated sequencing and three-dimensional imaging device may be usedfor detecting optical signals by fluorescence microscopy. According tocertain aspects, the fluorescence is generated by fluorescent dyes orfluorophores, such as cyanine. According to certain aspects, thefluorescence is generated by quantum dots or other types of nanoscalesemiconductors. According to certain aspects, the fluorescence isgenerated by fluorescent proteins, such as GFP. The automated sequencingand three-dimensional imaging device may be used for detecting opticalsignals of autofluorescence, chemiluminescence, or non-fluorescentoptical signals such as light absorption properties of a sample (e.g.,color) or light scattering properties of a sample, e.g. Ramanspectroscopy and CARS, coherent anti-Stokes Raman spectroscopy.

According to one aspect, an automated sequencing and three-dimensionalimaging device is provided which uses volumetric three dimensionalimaging modalities to image a three dimensional nucleic acid matrix. Theoptical configuration may be upright, inverted, side-view, dual-view,multi-view, or have any other orientation to enable measurement of lightsignals in three dimensions. The device includes hardware and softwarefunctionally assembled to enact a protocol of chemical manipulation(i.e., nucleic acid sequencing) and imaging of the three dimensionalnucleic acid containing matrix which is contained within a suitablevessel or stage. The device may be referred to as a fluidic sequencingmicroscope to the extent that it includes hardware and software toautomate sequencing and hardware and software for volumetric imaging.

The three-dimensional sequencing substrate may be contained within asample holder such as an enclosed flow cell, or be fully or partiallyexposed in an open well format. An enclosed flowcell may be taken tomean any solid or semi-solid substrate with an optically clear regionfor imaging and a channel in which laminar flow may be established forthe purposes of liquid exchange. The flowcell may have one more inletsor outlets or ports for the purposes of liquid exchange. These physicalinterfaces may be statically or dynamically coupled to a fluidics systemand may include wells or other reservoirs to store excess or reservefluidics to facilitate dispensing or extraction regimes. The sampleholder may be designed with additional physical features intended tointerface or cooperate with fluidics systems, optical systems, orphysical retention systems.

The device may include a stage for retaining the sample holder. Thestage may be positioned using high-precision motion control systems,such as linear servo motors with optical encoders. Optical encodersystems may be furnished in either absolute or relative formats. In thecase where the motor controller reads one or more relative encoders,homing routines may be implemented relative to limit sensors and/orphysical limits to provide repeatable axis positioning. Software limitsmay be dynamically imposed to prevent samples or sample holders (i.e.,flowcells) from colliding or interfering with other physical aspects ofthe device. Motor drivers responsible for the motor control mayinterface, over a standard communications protocol (e.g., RS-323,TCP/IP, UDP, etc), with other aspects of device software and hardwarefor the purposes of multi-axis, coordinated routines.

The stage may include physical mechanisms for positioning and retainingsample holders, such as slots with retention mechanisms (e.g., leafsprings or magnets) that reproducibly position sample holders/samplesfor interaction with fluidics and/or optical systems. The stage mayinclude a physical means of coupling fluidic apparatuses to sampleholders and/or samples. Stage components may be selected to minimize thetendency for thermal changes to introduce physical deformations ortranslations to the sample or sample holder. Stage components may beselected to minimize chemical reactivity with fluidics used duringchemistry protocols.

According to certain aspects, the device may include a mechanism forsample holder tracking, such as by reference IDs, barcodes, RFID tags,or inclusion of other trackable labels in the sample holder. Thetrackable label may be automatically detected by the device, such as bydetection of RFID or optical sensor for detecting a barcode, or thetrackable label may be input into a computer software system by a user.One or more types of sample holders may vary in physical organization ofthe samples, reagent input/output, imaging interface, and other aspectsof the sample holder. The sample holder tracking may further comprise amechanism for indicating to the device controller software theconfiguration of the sample holder, such as by referencing an index in adatabase of sample holder configurations. According to other aspects,the device may contain a mechanism for automatically configuring thesample holder configuration, such as by use of RADAR/LIDAR, includingRADAR/LIDAR on-a-chip systems, or by machine vision, wherein physicalaspects of the sample holder are automatically detected by the system,parsed, and used to configure the device interface with the sampleholder. According to other aspects, the device may utilize referencediagrams or fixtures for the purpose of configuring the device interfacewith the sample holder, such as a fixture for determining a physicaloffset for a feature of the sample holder.

According to certain aspects, the device also comprises a softwareinterface, such as a command line interface (CLI) or graphical userinterface (GUI) for configuring a sample holder definition or otherwiseconfiguring the interface between the device and the sample holder. Thedevice may automatically scan the imaging region of a sample holder forthe purpose of generating an overview scan of the image area within thesample holder. According to one aspect, the overview scan is presentedto the user via a GUI for the purpose of selecting regions of the samplefor sequencing and/or imaging. According to another aspect, the devicecontains software programs for automatically determining the appropriateregions of the sample for sequencing and/or imaging, such as bycalculation of a metric such as fluorescence intensity or entropy. Thedevice may further comprise a computer vision system or machine learningmechanism for feature recognition of the sample holder or componentsthereof.

The stage may include a heating or cooling apparatus where the stage maybe heated or cooled (such as through thermoelectric cooling usingPeltier elements), such as according to a programmed time andtemperature, such as is useful with thermo-cycling for applications suchas nucleic acid hybridization, amplification and sequencing. The heatingor cooling apparatus or heating or cooling unit may be capable of rapidtemperature cycling. The heating or cooling apparatus or unit may use aheat sink in conjunction with a fan to dissipate heat produced duringtemperature changes. The heating or cooling apparatus or unit may use aradiator and liquid cooling/circulating system to dissipate heatproduced during temperature changes. The heating or cooling apparatus orunit may use temperature sensors or thermistors as a means of providingtemperature feedback to a control system, which may be a microcontrolleror other electronic circuit.

The device may include a fluidics dispenser or fluidics unit whereprogrammed volumes of liquid reagents are dispensed to sample holders onthe stage, such as to wells or flow cells on the stage, and to thethree-dimensional nucleic acid containing matrix. The fluidics dispenseror unit may include temperature control (such as through thermoelectriccooling using Peltier elements) to store reagents at a consistenttemperature, and may implement either liquid or water cooling as a meansof heat dissipation. A pump, such as a syringe pump, may be used todeliver fluid reagents through the fluidics dispenser. According tocertain aspects, the fluidics and pressure-based fluidic dispense may bedriven by the pressure differential between the inside and outside of aliquid container. For example, a pressurized bottle may be connected toan electrically actuated valve, as by a piece of tubing, such that whenthe valve is opened, fluid is driven through the tubing and through thevalve onto a sample in an open well or into a closed flowcell. Incertain aspects, two or more reagents may be dispensed to a sampleholder from the same valve. These reagents may be selected upstream ofthe valve by means of another valve (e.g., a rotary valve) or otherphysical means. In other aspects, valves may be addressable by twoselectable lines, such that one line may deliver water or another liquidmeant to flush or otherwise clean the valve in between dispenses. Areservoir or absorbent material may be statically or dynamicallypositioned beneath the valve to collect rinse solution or reagent duringpriming and cleaning operations.

In certain implementations, the exchange of liquids in a closed flowcellmay take place in an entirely closed-loop system, where tubing isconnected to the flowcell in a fixed manner and a system of valves andpumps (or other pressure generating devices) controls the movement andselection of reagents delivered to the flowcell. Closed flowcells may bedesigned to allow for non-contact reagent dispensing into wells externalto the sample channel. Such wells may be designed to hold a volumeappropriate to partially or fully displace the volume of thesample-containing channel. The movement of dispensed reagent into theflowcell may then be driven by positive pressure exerted on the wells,or by negative pressure exerted on outlets at the opposite end of thesample channel. The interface between the suction-generating device andthe flowcell may be transient or fixed, and may be created or adjustedby motion axes such as those driven by stepper motors, servo motors orsolenoids. The interface may be further defined by the presence ofsuction cup devices designed to create and maintain a consistent sealwhile minimally perturbing the physical positioning of the sample.

In other aspects, sample holders with open well(s) may be designed withfeatures to accommodate reagent dispensing and extraction to minimizesample perturbations, such as by including channels, slots, or otherphysical areas designed to sequester higher levels of liquid flow fromthe sample. The open well(s) may additionally have chamfered orotherwise tapered sides to accommodate the specific shape of opticalsystem interfaces, such as by matching the side profile of an opticalobjective.

According to a certain implementation of the pressure-driven fluidicsblock, multiple distinct tubes of reagent sit inside a pressurizedcontainer, each connected to its own valve, such that when any one ormore valves are actuated fluid is driven from the tube through the valveonto the sample. The valves may be of one or more general types,including but not limited to solenoid valves, rotary valves andmicrofluidic valves. The valves may be select to maximize precisionand/or minimize dead volume. According to certain aspects, the valvebodies may be designed in such a way as to place the actuating mechanismas close to the orifice as possible, to minimize problems associatedwith reagent drying and/or clogging. The valves may have upstreamfilters, screens, or other means of preventing non-liquid materials fromclogging or otherwise interfering with the valves' operation. The amountof reagent dispensed using such a pressure-driven system is determinedbased on the valve internal diameter, liquid viscosity, pressuredifferential, and time of valve actuation, and can be determinedempirically and used to configure a software controller such that adesired volume is converted into a length of time (given a particularhardware configuration with particular liquid viscosity, valve internaldiameter, and pressurization) during which the valve is opened oractuated. According to certain aspects, the reagent containers may beindividually or collectively pressurized. The pressurization medium isideally an inert gas such as argon. The fluidic systems may combinepump-driven and pressure-driven fluidic components. The device mayinclude a mixer for mixing programmed volumes of liquid reagents. Theaspiration system described herein is used to remove fluid from a flowcell by applying a suction force to the liquid using a pump or vacuumsource. Pressure-based dispense may also be used to exchange fluidsduring sequencing by introducing a new reagent under pressure as aboveto displace the existing fluid through a channel. Pressure baseddispense may be used alongside aspiration to exchange liquid in a flowcell.

According to one aspect, the device may contain a reservoir forcontaining bulk reagents, or reagents with larger volume. According toone such aspect, the bulk reagent reservoir may contain one or morepressurized containers within an enclosing container. According toanother such aspect, the bulk reagent reservoir may contain one or morecontainers within a pressurized enclosing container. According to oneaspect, the bulk reagent reservoir is temperature controlled, such asthrough thermoelectric cooling using Peltier elements, for the purposeof extending the life of and activity of one or more reagents. Theheating or cooling apparatus or unit may use a heat sink in conjunctionwith a fan to dissipate heat produced during temperature changes. Theheating or cooling apparatus or unit may use a radiator and liquidcooling/circulating system to dissipate heat produced during temperaturechanges. The heating or cooling apparatus or unit may use temperaturesensors or thermistors as a means of providing temperature feedback to acontrol system, which may be a microcontroller or other electroniccircuit. The bulk reagent reservoirs may be connectorized, such as bysplit septum, enabling facile exchange of individual reservoirs. Otherconnector types include, but are not limited to, Luer locks, ¼ 28,MINSTACK (LEE Co).

The device may include a mechanism of extracting liquid from a flowcell. According to one aspect, a vacuum is generated by a pump, such asa main pump or syringe pump, which is used to aspirate liquid containedin a flow cell, such as an open well or partially enclosed flow cell.

According to one aspect, a motorized sipper tube is used to contact thesample holder or liquid therein for the purpose of extracting liquid.Further according to this aspect, the motorized sipper tube may comprisea pneumatic, servo, or stepper motor, a positional encoder, andelectromechanical controller system. The device may contain one or morewaste liquid repositories, into which waste or extracted liquid isdirected for the purpose of temporary storage. Waste liquid may bedirected by means of pumps and valves, such as a rotary valve. The wasteliquid repository may comprise a feedback system for automaticallynotifying a user upon reaching a certain level. The waste liquid may bedirected to one or more repositories based on the nature of the reagent,to prevent undesired chemical reactions, or to facilitate downstreamdisposal according to applicable procedures, laws, or regulations.

The device may include a mechanism of covering one or more flow cells,wells, sample holders and/or stages for the purpose of preventingaccumulation of airborne particulates in the sample and/or preventingevaporation of liquid reagents. According to one aspect, the devicecontains a motorized flowcell cover that uses one or more motion axes toposition an airtight cover over one or more flow cells, wells, sampleholders and/or stages. According to another aspect, the stage contains adynamically extendable cover. According to another aspect, the devicecontains a static cover capable of interfacing with the motorized stagefor the purpose of enclosing the wells and/or sample holders.

The device may include an optical assembly including one or more opticalaxes. The device may include one or more detectors, such as large areadetectors, for volumetric imaging of the three dimensional nucleic acidcontaining matrix. The detectors may be cameras, and in particular,cameras with physical attributes tailored to high-speed, low-noisescientific imaging, such as an sCMOS camera. In one such embodiment, areflection based autofocus system provides closed loop control of theoptical axis in order to attain and/or maintain sample focus. In anotherconfiguration, a microcontroller, FPGA, or other computing device mayprovide software focus and positioning feedback using one or more imageanalysis algorithms such as that described in Mario, A. Bueno, JosueAlvarez-Borrego, and L. Acho. “Autofocus algorithm using one-dimensionalFourier transform and Pearson correlation.” 5th Iberoamerican Meeting onOptics and 8th Latin American Meeting on Optics, Lasers, and TheirApplications. International Society for Optics and Photonics, 2004hereby incorporated by reference in its entirety. Such automated samplepositioning may include coordination of one or more motion axes inconjunction with the imaging system. The sample positioning may accountfor physical shifts in sample position over the course of imaging andmay be tolerant of shifts that are greater than the field of viewcaptured in a single image frame. The device may implement a means ofmapping a planar surface, rendering the need for autofocusing in realtime unnecessary. Such a system may include using a reflection- orsoftware-based autofocus system to sample three or more points at thesample surface and then fitting those points to the equation for a planeor the surface geometry of the solid substrate. The fitting process mayinvolve excluding one or more points based on autofocus signal data, itsresidual as a result of regression analysis, or other factors indicatingits fitness as a data point. The fitting process may include allowancesfor surface variance consistent with the sample mounting medium, suchthat the final surface map may include local deviations from a perfectlyflat plane to reflect variations in the actual substrate surface. Asdescribed in FIG. 12, according to one aspect the device comprises asystem for mapping the surface of a sample holder with respect to one ormore motion axes for the purpose of reproducible positioning of thesample relative to the imaging system, comprising the steps of using: 1)an autofocus system, such as a laser-reflection autofocus mechanism, toacquire one or more distance measurements between the optical system andthe sample, and 2) computing one or more offsets relative to thepositional encoder(s) along the one or more motion axes, which are usedduring image acquisition.

The device may use image-based software programs for determining,adjusting, correcting, and/or tracking the position of the sample.According to one aspect, image data is computationally registered to areference for the purpose of calculating a positional shift along one ormore dimensions. Further according to this aspect, a Fourier transform(FT), such as the discrete Fourier transform (DFT) or fast Fouriertransform (FFT) is used to compute a shift between two or more images orimage volumes along one or more dimensions. According to one aspect,sub-pixel shifts are calculated, such as by using the upscaled DFT.According to one aspect, translational shifts are calculated along oneor more dimensions. According to another aspect, rotational shifts arecalculated along one or more dimensions. According to one aspect,features or fiducial markers contained within the sample holder,flowcell, well, or sample, are used for the purpose of aiding positionaltracking using image analysis. Features or fiducial markers includefeatures manufactured into or added onto the sample holder, flowcell, orwell, such as engraved features, laser-engraved features, printedfeatures, deposited features, microcontact printed features, beads, andother types of patterns in one or more dimensions. According to oneaspect, fiducial markers are embedded into the sample, such as into the3D hydrogel. According to one aspect, the fiducial markers aremicroscopy beads, which may be fluorescent or autofluorescent. Accordingto one aspect, features are an aspect of one surface of the solidsubstrate forming part of the sample holder. According to a certainaspect, the features are an aspect of a different surface of the sampleholder than the surface containing the sample.

In one example, the flowcell is understood to be a glass slidecontaining two or more open wells on the top surface, with beads servingas fiducial markers deposited on either the top or bottom surface of theglass slide.

A microcontroller system coordinates motion systems in XYZ axes alongwith the illumination/excitation light source and camera sensor suchthat the motion systems position the sample relative to the opticalsystem, then image data is acquired at that position with global shuttercapture or rolling shutter or “all lines firing” rolling shutter captureusing synchronized illumination. In other cases, the motion along one ormore axes are synchronized with the capture of lines along the camerasensor such that the sample does not need to come to a resting positionrelative to the optical system. Software and hardware handshakingprotocols are implemented over low-latency communication protocols(e.g., digital TTL, I2C, RS-232, UDP, TCP/IP) to coordinate between thesubsystems, e.g. initiating axis motion or triggering a camera exposure.

The device may include one or more objective lenses for imaging. In acertain aspect, the device may include one objective lens. In anotheraspect, the device may contain two objective lenses. In another aspect,the device may contain three or more objective lenses. The objectivelenses may be water immersion lenses, oil immersion lenses, waterdipping lenses, air lenses, lenses with a refractive index matchinganother imaging medium, or lenses with an adjustable refractive index.In a certain aspect the device contains a single water dipping objectivelens, which provides for higher image quality by eliminating therefractive index mismatch occurring at the interface between two mediawith distinct refractive indexes, such as an air-water interface orwater-glass interface.

In aspects comprising one or more objective lenses with refractive indexnot matched to air, the objective lens must interface with an imagingmedia, such as water, oil, or other imaging buffer. In these aspects,the device may comprise a mechanism of wetting the objective lenses orotherwise creating an interface between the objective lens and theimaging medium. Certain mechanisms of lens wetting include dipping thelens into an imaging medium or otherwise dispensing an imaging mediumonto the lens, such as by a syringe, needle valve, or other mechanismsknown to those familiar with the art of dispensing a liquid reagent. Ina certain aspect, the device dispenses a certain amount of liquid into awell for the purpose of creating an incident angle between the objectivelens and the liquid interface for deposition of liquid onto theobjective lens without forming bubbles. In another aspect, the devicedispenses a certain amount of liquid onto the objective, such as byusing a syringe or needle valve, without forming bubbles, as bycontrolling the speed and angle of incidence between the liquid dropletand the objective lens.

During imaging in a liquid imaging medium, bubbles may form either onthe objective lens, within the sample, or between the objective lens andthe sample; including in devices imaging within an open flowcell andthrough a glass interface present in a closed flowcell. The device maycontain a mechanism of detecting bubbles formed on the objective lens,or bubbles present between the objective lens and the sample. Mechanismsof bubble detection include detection via scattering of light; by imageanalysis, e.g., by measurement of the point spread function of theoptical system, which is perturbed by bubbles; by external machinevision, such as by a camera or other imaging system observing the lens,connected to a computer system with software programmed to detectbubbles on the lens. The device further may contain a mechanism foreliminating bubbles formed on the objective lens. According to oneaspect, the device comprises a mechanism for contacting the objectivelens with an aspirating needle, which removes any liquid present on theobjective lens. According to another aspect, the device comprises amechanism for contacting the objective lens with an absorbent material,which absorbs any liquid present on the objective lens. The device maycontain a mechanism for drying or removing liquid from the objectivelens. The device may execute a software and hardware routine forremoving and replacing a liquid imaging medium from the lens upondetection of a bubble. The device may contain a mechanism for alerting auser upon detection of bubbles on the objective lens, within the sample,or between the objective lens and the sample.

During operation of the device, the objective lens may accumulate dirt,dust, deposited salts or other reagent solutes, or other types ofmaterials which interfere with imaging. The device may contain amechanism for detecting such interference, via scattering of light orother properties of the interaction between light and the interferingmaterial; by image analysis, e.g., by measurement of the point spreadfunction of the optical system, which is perturbed by the presence ofinterfering materials; by external machine vision, such as by a cameraor other imaging system observing the lens, connected to a computersystem with software programmed to detect interfering materials on thelens. The device may further contain a mechanism for cleaning theobjective lens. According to one aspect, the device may include a lenscleaning reagent, which is dispensed onto the lens for the purpose ofcleaning the lens, such as by a syringe or needle valve, or by dippingthe objective lens into a cleaning reagent dispensed into a well or ontoa non-abrasive material, which is made to contact the objective lens.The device may contain a mechanism for alerting a user upon detection ofan interfering material on the objective lens, within the sample, orbetween the objective lens and the sample.

The device comprises one or more optical light paths. The device maycontain optics for the purpose of correcting refractive index mismatchesbetween certain components of the optical system and the sample orimaging medium. The device may contain optics for the purpose ofcorrecting other types of optical distortion within the optical system,such as spherical or chromatic aberration. The device may contain amechanism of detecting optical distortion, such as by using an imagesensor combined with software to detect changes in a point-spreadfunction of the optical system or other property of the optical system.According to one aspect, the device comprises one or more beamcharacterizing cameras. According to another aspect, prior to, during,or after operation of the device, the device may contain an automated ormanual routine for measuring the point spread function or other opticalproperty of the system and alerting a user or engaging a mechanical,electromechanical, or optical system for correcting the opticaldistortion. According to a certain aspect, the device contains anadaptive optical system (AO), which is used to improve the performanceof optical systems by reducing the effect of wavefront distortions.Adaptive optics can correct deformations of an incoming wavefront bydeforming a mirror in order to compensate for the distortion. Theadaptive optics system may comprise a deformable mirror, image sensor,and hardware and software feedback systems. According to one aspect, theadaptive optic system contains a wavefront sensor, such as theShack-Hartmann wavefront sensor. The adaptive optical system and othercorrective optical systems may be open loop, where errors are measuredbefore they have been corrected by the corrector. The adaptive opticalsystem and other corrective optical systems may be closed loop, wherethe errors are measured after they have been corrected by the corrector.Adaptive optics may be used to improve the image quality within a 3Dsample by correcting for optical aberrations within the sample.

The device may include one or more electromechanical, electronic, orfully computerized systems for the purposes of controlling andcoordinating the timing of fluidic, optical, and motion-related events.Device subsystems such as motor controllers, temperature controllers,pneumatics controller, valve controllers, cameras, optical tuning orgating systems, sensors, and other electronic systems may leverage avariety of communication protocols for the purposes of suchcoordination. Communication protocols may be selected on the basis oflatency, interoperability, electromechanical constraints or otherapplication-focused considerations. Subsystems may conform to consistentor well-defined application program interfaces (APIs) such that they maybe individually addressed and/or operated from generic computers orhuman machine interfaces.

Timing of optical systems such as cameras, confocal optical systems,illumination devices, AOTFs, mechanical shutters, etc, and single- ormulti-axis motion control systems may be coordinated bymicrocontrollers, motor controllers, electronic circuits, and/orcomputerized systems. Optical sensor exposure timing may be optimizedsuch that motion control movements overlap with non-measurement sensorevents such as pixel readin/readout or background measurements. Opticalillumination timing (e.g., laser illumination gated) may be implementedsuch that it is tied to specific optical sensor events (e.g.,readin/readout) so as to minimize sample exposure to excitation light.Single- or multi-axis motion control for the purposes of optical imagingmay be further optimized to account for the sensor exposure regimen(e.g., rolling shutter exposure) for continuous motion applications.Under certain specialized imaging regimes, e.g., time delay integration(TDI), it is possible to execute continuing axis motion during theacquisition of imaging data.

In certain aspects, the dimension order of multi-axis motion control(when coupled with optical imaging) may be selected so as to minimizeframe-to-frame move times. For example, it is often the case thevertical axis (i.e., Z or optical axis) move times are the fastest, soit is desirable to perform three-dimensional imaging in a series of “Zstacks” in which frames are acquired while the vertical axis is drivenup or down. These Z stacks are performed repeatedly across an X/Y planeor plane-like surface in order to acquire a fully three-dimensionalvolume of image data.

In some cases, it may be desirable to image a three-dimensional volumethat is not cuboid in nature. In these cases, a three-dimensionalcoordinate system based on physical or engineering units may be employedto dictate arbitrary three-dimensional imaging positions that are thendisseminated to the participating motion control and imaging systems.Employing such a system allows for imaging that is constrainedexclusively to the region of a three-dimensional matrix in which samplevoxels of interest exist.

It may be desirable to image the volume with a particular spatialsampling frequency. In certain aspects, the sampling frequency ofimaging along one or more axes is determined relative to the Nyquistfrequency of the optical system. In a certain implementation, image dataacquired using a 40×1.0 NA objective is sampled in the axial (Z) axis atapproximately 500 nanometer intervals. In another aspect, the samplingfrequency of imaging along one or more axes is oversampled, such as byacquiring image data or sampling the same area of the sample volumetwice. Further according to this aspect, oversampling the volume mayfacilitate computational volumetric reconstruction, such as by providingredundant image data in neighboring image frames for the purpose ofvolumetric image stitching. In a certain implementation, image data maybe acquired with 10% overlaps along one or more axes, with 20% overlapsalong one or more axes, or with 30% or more overlapping pixel data alongone or more axes.

The device may include more than one stage, fluidics unit, and/oroptical system such that each sample holder and/or sample may beaddressed by multiple systems. Under such a regime access to certainhardware resources must be shared and therefore a physical resourceallocation and scheduling system is necessary. Such a scheduling systemmay be passive or active, and may include predictive modeling as a meansof anticipating and scheduling hardware resource access based on futurefluidic and optical imaging needs on a per-sample basis. Further aspectsof the scheduling system may allow for external events, such as thoseperformed by a user or an external computer system to interrupt orotherwise modify the activities of the fluidic imaging system. In onesuch aspect, a physical media storage system may become full and nolonger able to store acquired images. This event could trigger acessation of imaging activities on the device pending external userintervention. In another aspect, a user may want to add a sample in asample holder to a slot on a device stage. The user may be able to,through an HMI, graphical user interface, or using a physical button,temporarily pause or otherwise modify the operational state of thedevice to allow for the addition of the sample holder.

The device may be operated and otherwise programmed through a commandline interface, graphical user interface, or application programminginterface (API) exposed through local software libraries over a networkconnection. The user may be able to perform activities including but notlimited to: physical configuration of the device, testing andcalibration tasks, sample holder and sample definition generation andmodification, fluidic manipulations and routine specification, opticalmeasurement and routine specification, sequencing protocol development,as well as other tasks related to general fluidics, optical imaging, andmotion control. The device may include hardware or software that enablesreal time analysis of imaging data, such as FPGAs or user-space softwarerunning on GPUs that allows for activities such as three-dimensionalimage alignment, objective finding, signal processing or measurement,and other image analysis tasks as required by sequencing and opticalsample analysis. The device may include hardware and software designedto store image data locally. The device may be part of a network ofdevices and services designed to collect, analyze, store, and visualizethree-dimensional imaging and sequencing data. This network may includephysically co-located hardware as well as cloud computing faculties. Incertain embodiments it may implement open standards to interface withthird party hardware and software.

It is to be understood that the device includes software to automate theprocedures of amplification and sequencing as described herein. It is tobe further understood that the hardware components described herein arecommercially available in whole or in part. According to one aspect, acommercially available sequencing apparatus called the “I15 Pollinator”manufactured by Danaher Corporation may be used for the sequencingaspects described herein and may be modified to include the hardware forvolumetric imaging described herein.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference in their entirety forall purposes.

Example I Operative Components of an Automated Sequencing andThree-Dimensional Imaging Device

FIG. 2 is a perspective view in schematic of operative components of anautomated sequencing and three-dimensional imaging device of the presentdisclosure. It is to be understood that further components may be addedto the device including housing elements. As shown in FIG. 2, theautomated sequencing and three-dimensional imaging device includes alaser confocal head enclosure including a pinhole array and cameradetectors for receiving light from the sample. Such a hardwarearrangement allows for volumetric imaging of a three dimensional nucleicacid containing matrix. The device further includes an optical path withreflection-based autofocus to the laser confocal head enclosure. Thedevice further includes a temperature controlled fluidics head on apressurized fluidics block which serves to provide one or more reagentsto the sample. Operatively connected bulk fluidics valves and anaspiration tube for removing liquid from the sample are provided. Asample stage with temperature control and XY motion system is providedand is shown with a glass slide sample holder. A track for stage motionwith linear encoders is operatively connected to the stage. An enclosuredoor for an oxygen-free (argon) environment is provided with the rest ofenclosure not shown. The device rests on an anti-vibration foundation.

FIG. 3 is a front view in schematic of operative components of anautomated sequencing and three-dimensional imaging device of the presentdisclosure. The device includes an optical path including laserreflection-based autofocus and a laser confocal head. A bulk reagentvalve array for bulk reagent dispense is shown (pressurized bulk reagentstorage not shown). A Peltier element for reagent block temperaturecontrol and heat sink (shown) or liquid cooling is shown connected to afluidics block for pressure-based fluidics dispense. A dippingaspiration tube for removing liquid from the flow cell is shown.Electronics are provided for valve actuation and fluidics blocktemperature and pressure control and feedback. A valve array is shownconnected to the fluidics block. A stage for holding samples, withtemperature control by Peltier element and heat sink (shown) or liquidcooling is provided. Electronics for stage motion and temperaturecontrol and feedback are provided. Track for stage motion with linearencoders for positional feedback is provided. An optical axis withmicroscope objective is shown. A dipping objective is shown for openflowcell. An enclosure door for oxygen-free (argon) environment isprovided (rest of enclosure not shown). The device includes a vibrationcontrol rigid foundation. As can be seen in FIG. 3, the device forvolumetric imaging of a 3D sequencing matrix utilizes two independentstages and two independent fluidics systems along with a single opticalaxis for image acquisition. Image acquisition is in practice thetemporally limiting step. No matter how long the biochemistry andfluidics for sequencing take per base, this is a fixed time per base(e.g. 2 hours), but the imaging time is proportional to the size of thevolume being imaged. By using two stages, each operating independentlyand with independent fluidics, one stage can be doingbiochemistry/fluidics while the other stage is imaging. Therefore thesystem is always effectively waiting to image and the fixed biochemistrytime is effectively zero as the system is never waiting on fluidics tocomplete in order to proceed to the next step, but is always waiting onimaging to complete. Scan time may be an issue for 3D optical scanningvs. 2D optical scanning, as 2D scan time increases to the second powerwith scanning area, but in 3D scan time increases to the third powerwith scanning volume. The configuration of two independent stages andtwo independent fluidics systems along with a single optical axis forimage acquisition is also cost effective, as the stage withmotion/temperature control stage and fluidics subsystems are relativelycheap compared to the optical subsystem, which requires costly optics,camera detectors, data acquisition electronics (e.g. frame grabberhardware to capture image data from the cameras), and laser sources forfluorescent excitation.

FIG. 4 is a perspective view of the device of the present disclosure.Laser scanning confocal head and camera detectors are provided. Anoptical path light bend into the confocal head is provided. A microscopeobjective is shown, e.g. a dipping immersion objective for perfectlymatched refractive index with the sample for high fidelity imaging. Amirror for capturing feedback light signal of reflection-based autofocussystem is provided. An open flow cell is shown inserted into the stage.A Z-axis linear motor with linear encoder feedback for Z motion and Zscanning of objective focal plane through a sample is provided. Locatedbehind the Z-axis linear motor is the reflection-based autofocus system(not visible). A pressurized and temperature controlled fluidic block isshown. A valve for fluidic dispense is provided. Tubes holding reagentsfor dispense are shown inside the pressurized fluidics block.

FIG. 5 is a perspective view of a stage according to the presentdisclosure. A heat sink for the Peltier cooler for stage temperaturecontrol is provided. A flow cell foundation is shown as a standard 1×3inch 1 mm thick glass microscopy slide onto which the 3D sequencinglibrary matrix is attached. The 3D sequencing library matrix attachedinside the well. A fluidic well component of the open-format flowcell isshown as a 2-well format. An open-format flowcell avoids problems withbubbles and laminar flow over irregular 3D matrix that would causeuneven diffusion of reagents into and out of the 3D sequencing librarymatrix. A spring-loaded registration point for reproducible slidepositioning is provided. The flow cell contacts a thermo-cycling stagetop. A rail system for reproducible slide positioning is provided thatprovides 2D directional force onto the slide (downward and along one ofthe long edges of the slide) and secures the sample. A registrationpoint for reproducible slide positioning is provided. In oneimplementation, the flow cell consists of a series of open-chambers orwells. Reagents are dispensed into the wells from the top by the fluidicsystem and removed by an aspiration tube.

Open well format is preferable for 3D matrix samples as formats enclosedon top and bottom may suffer from issues related to fluid dynamics suchas uneven laminar flow over an irregularly-shaped 3D matrix, andintroduction or trapping of bubbles by vortexes created in the dynamicliquid field. Open well format is amenable to upright (from the top)microscopy modalities by one or more optical axes where the objectivephysically dips into an imaging medium such as an imaging buffer,minimizing or eliminating refractive index mismatches between theoptical system and the sample (e.g. air/glass interface).

FIG. 6 is a schematic timing diagram illustrating an example ofcoordination between optical imaging, illumination, motion control, andacquisition systems for the purposes of achieving optimal imaging framerates.

FIG. 7 is a schematic block diagram illustrating an example TTLcommunication structure for the purposes of optimal real timecoordination of optical imaging, illumination, motion control, andacquisition systems.

FIG. 8 depicts the timing and resource usage timing diagram forsequencing device fluidics, imaging, and dual stage subsystems. Thisfigure depicts a device with two independent motion stages, which housethe sample holders, two intendent fluidic systems, denoted “Left” and“Right”, and a single imaging axis. A controller manages the motionstages such that at Time=0, both stages are engaged in fluidic routines.At Time=1, image data is acquired from the samples on Stage 1 whileStage 2 is engaged in fluidic routines. At Time=2, image dataacquisition from Stage 1 has completed, and fluidic routines for Stage 2have completed. At Time=3, image data is acquired from the samples onStage 2, while Stage 1 is engaged in fluidic routines. At Time=4, imagedata acquisition from Stage 2 has completed, and fluidic routines forStage 1 have completed.

FIG. 9 depicts a schematic block diagram illustrating the controllerorganization for the dual stages, fluidic, and imaging subsystems. Acontroller synchronizes event timing, such as that depicted in FIG. 8.

FIG. 10 depicts device systems for managing the state of and interfacingwith an objective lens. Subpanel 100 is directed to depiction of a dryobjective (105) and a wet objective (110) comprising the objective lenswith a droplet of imaging medium (115). Imaging medium include aqueousimaging buffers, such as water and anti-fade reagents, as well as oiland other types of imaging liquids. Subpanel 200 depicts a configurationof an apparatus for objective wetting and drying, such as an apparatusfor removing a bubble from an objective lens or for cleaning anobjective lens; a carrier is depicted (220), which may be a fixture ofthe device or a consumable insert, which further comprises a modality(230) for depositing a reagent (235) onto the objective lens, such as awell or reagent dispensing apparatus, and also further comprises amodality (240) for removing liquid from an objective lens, such as anon-abrasive absorbent material or aspirating port. Subpanel 300 depictsa routine for depositing liquid onto an objective lens, comprising thesteps of forming a liquid interface on the liquid wetting modality(310), contacting the objective lens with the liquid interface (320), asby translating the objective lens or objective wetting modality, suchthat the objective is wetted (330). Subpanel 400 depicts a routine forremoving liquid from an objective lens (410), comprising the steps oftranslating the objective lens or drying modality such that the dryingmodality contacts the liquid forming an interface (420) such that theliquid is absorbed or otherwise aspirated by the drying modality,resulting in an objective lens without a liquid droplet (430).

FIG. 11 depicts a flow chart for repeatable XYZ positioning of thesample relative to the imaging axis over time. In the case where thesolid substrate may shift over time, this system for determining shiftsof a sample holder can increase the accuracy of XYZ positionalrepeatability over time between the sample and the imaging system. Atthe initialization of imaging, the device moves Position 1 under theimaging axis and acquires initial image data, which is transmitted to acomputer system. The Computer system computes a physical offset relativeto a reference image dataset, which is communicated to the SequencingDevice and stored. As depicted in Loop 510, the sequencing device mayrepeat these steps until the physical offset received by the computersystem is lower than a threshold amount, e.g., until the computer systemconfirms the physical positional information stored by the devicecorresponding to Position 1 is within a given physical shift relative toa reference positional state.

FIG. 12 depicts a system for mapping a surface of a sample holdercomprising a solid substrate. In the case where the solid substrate mayshift over time and a particular autofocus system may have a certainfailure rate, this system for mapping the surface of a sample holder canincrease the accuracy of Z positional repeatability over time betweenthe sample and the imaging system, such as by discarding outliers in theZ offsets as determined by the autofocus system. This system may alsoincrease image acquisition speed, as pre-computing physical offsets forthe motion system may be significantly faster than allowing theautofocus to determine the physical offset in real time at eachpotential imaging position. Panel 600, which includes Subpanels 610,650, and 700, depicts a diagram of the device state. In Subpanel 610, anobjective lens (620) is positioned relative to a solid substrate (630),such as glass, silicon, metal, plastic, or another solid material, whichis understood to immobilize a mounted sample such as a FISSEQ hydrogel.This depiction includes a laser-based autofocus device, which shines alaser (640) onto the solid substrate in order to determine the distancebetween the objective and the solid substrate, such as by analyzing thereflection of the laser light from the surface. Subpanel 650 depicts aside view of subpanel 610, wherein the distance 680 between theobjective and the solid substrate corresponds to a known value, such asthe focal length of the objective, when the objective lens is determinedto be focused on the solid surface. Subpanel 700 depicts scanning theobjective (710) in one, two, or three axes (715 depicts three possiblemotion axes) over the solid substrate (720), such that an Z autofocusoffset is measured at each scan position (730). Subpanel 900 depicts aflow chart for the behavior of the device and a computer system duringsurface scanning, wherein the objective lens is positioned at positionk, and a Z offset is determined and stored, as in an array. Uponsampling a number of positions, the Z offsets are communicated to acomputer system, which computes physical offsets, such as by fitting theautofocus offsets to a plane. The physical offsets are communicated backto the sequencing device, which proceeds to acquire 3D imaging data withaccurate Z physical offsets. Subpanel 800 depicts a diagram of autofocusZ offsets being fit to a plane for the purpose of determining accuratephysical Z offsets.

The practice of the methods disclosed herein may employ conventionalbiology methods, software, computers and computer systems. Accordingly,the methods described herein may be computer implemented methods inwhole or in part. Computer software utilized in the methods of thepresent disclosure include computer readable medium havingcomputer-executable instructions for performing logic steps of themethod of the invention. Suitable computer readable medium include, butare not limited to, a floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive,flash memory, ROM/RAM, magnetic tapes, and others that may be developed.The computer executable instructions may be written in a suitablecomputer language or combination of several computer languages. Themethods described herein may also make use of various commerciallyavailable computers and computer program products and software for avariety of purposes including obtaining and processing light intensityinto data values, automation of reagent delivery, automation of reactionconditions such as thermo-cycling, automation of movement of a stageincluding a sample holder and a sample and processing and storage oflight intensity data and other methods and aspects described herein.

Aspects of the present disclosure are directed to a method of analyzinga plurality of nucleic acids within a three dimensional polymerizedmatrix including amplifying the plurality of nucleic acids to produceamplicons within the matrix, covalently bonding the amplicons to thematrix, sequencing the plurality of amplicons using an opticalsequencing method where the plurality of amplicons are labeled with adetectable label, and volumetrically imaging the plurality of ampliconsto produce three dimensional imaging data of the plurality of ampliconswherein light intensity data is processed into a three-dimensionalvolumetric image. According to one aspect, the plurality of nucleicacids is contained within a biological sample and the matrix-formingmaterial is introduced into the biological sample. According to oneaspect, the plurality of nucleic acids is contained within a cell andthe matrix-forming material is introduced into the cell. According toone aspect, the plurality of nucleic acids is contained within a tissuesample and the matrix-forming material is introduced into the tissuesample. According to one aspect, the three dimensional imaging dataidentifies the relative position of the plurality of amplicons withinthe cell. According to one aspect, the plurality of amplicons issequenced using fluorescence in situ sequencing. According to oneaspect, the plurality of nucleic acids are volumetrically imaged usingone or more of 3D structured illumination, selective planar illuminationmicroscopy, light sheet microscopy, emission manipulation, volumetricimaging using pinhole confocal microscopy, volumetric imaging usingaperture correlation confocal microscopy, volumetric imaging usingvolumetric reconstruction from slices, volumetric imaging usingdeconvolution microscopy, volumetric imaging using aberration-correctedmultifocus microscopy, volumetric imaging using digital holographicmicroscopy.

Aspects of the present disclosure are directed to an automatedsequencing and volumetric imaging device including a multi axis stage orpositioning system including a sample holder for a three dimensionalnucleic acid containing matrix, a heating or cooling apparatusoperationally connected to the stage, whereby the heating or coolingapparatus is programmable for time and temperature useful withthermo-cycling for amplification and sequencing, a fluidics dispenserpositioned to dispense one or more reagents into the sample holderwherein the fluidics dispenser is in fluid communication with one ormore reservoirs for containing one or more reagents, whereby thefluidics dispenser is programmable for dispensing programmed volumes ofliquid reagents to the sample holder, a pump operationally connected tothe fluidics dispenser whereby the pump forces or withdraws one or moreregents from the one or more reservoirs through the fluidics dispenser,an optical assembly including one or more optical axis, one or moredetectors positioned in light receiving communication with the sampleholder, whereby the one or more detectors receives light intensitysignals which processed into a three-dimensional volumetric image of thenucleic acid sample, and one or more microprocessors with software forautomating and controlling introduction of reagents into the sampleholder, thermocycling of the sample holder, and image detection andacquisition.

REFERENCES

Each reference is incorporated herein by reference in its entirety forall purposes.

-   Drmanac, R., Sparks, A. B., Callow, M. J., Halpern, A. L., Burns, N.    L., Kermani, B. G., Carnevali, P., Nazarenko, I., Nilsen, G. B.,    Yeung, G., et al. (2010). Human genome sequencing using unchained    base reads on self-assembling DNA nanoarrays. Science 327, 78-81.-   Islam, S., Kjallquist, U., Moliner, A., Zajac, P., Fan, J. B.,    Lonnerberg, P., and Linnarsson, S. (2011). Characterization of the    single-cell transcriptional landscape by highly multiplex RNA-seq.    Genome Res 21, 1160-1167.-   Larsson, C., Grundberg, I., Söderberg, O., and Nilsson, M. (2010).    In situ detection and genotyping of individual mRNA molecules.    Nature methods 7, 395-397.-   Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A. K.,    Landegren, U., and Nilsson, M. (2004). In situ genotyping individual    DNA molecules by target-primed rolling-circle amplification of    padlock probes. Nature methods 1, 227-232.-   Shendure, J., Porreca, G. J., Reppas, N. B., Lin, X., McCutcheon, J.    P., Rosenbaum, A. M., Wang, M. D., Zhang, K., Mitra, R. D., and    Church, G. M. (2005). Accurate multiplex polony sequencing of an    evolved bacterial genome. Science 309, 1728-1732.

What is claimed is:
 1. A method for nucleic acid analysis, comprising:(a) providing a biological sample comprising a plurality of nucleic acidmolecules in a three dimensional (3D) matrix, which plurality of nucleicacid molecules have a relative 3D spatial relationship; (b) subjectingsaid plurality of nucleic acid molecules to nucleic acid amplificationunder conditions sufficient to form a plurality of amplicons within said3D matrix, wherein subsequent to formation, said plurality of ampliconsare coupled to said 3D matrix and have said relative 3D spatialrelationship; (c) subjecting said plurality of amplicons to nucleic acidsequencing while said plurality of amplicons are coupled to said 3Dmatrix, which nucleic acid sequencing comprises (i) sequentiallycontacting said plurality of amplicons with detectable labels and (ii)using a detector to obtain signals corresponding to said detectablelabels from said 3D matrix, which signals are obtained by said detectorfrom a plurality of planes during said nucleic acid sequencing; and (d)using said signals obtained by said detector from said plurality ofplanes in (c) to generate a 3D volumetric image of said plurality ofamplicons, which 3D volumetric image identifies said relative 3D spatialrelationship of said plurality of amplicons.
 2. The method of claim 1,wherein (b) further comprises coupling said plurality of amplicons tosaid 3D matrix.
 3. The method of claim 1, wherein subsequent toformation, each of said plurality of amplicons is covalently coupled tosaid 3D matrix.
 4. The method of claim 1, further comprising, prior to(a), (i) introducing a matrix-forming material into said biologicalsample, and (ii) using said matrix-forming material to generate said 3Dmatrix.
 5. The method of claim 1, wherein said biological sample is acell.
 6. The method of claim 5, wherein said 3D matrix is in a cell, andwherein in (c) said signals are obtained from within said cell.
 7. Themethod of claim 6, wherein said signals are fluorescence signals.
 8. Themethod of claim 1, wherein said biological sample is a tissue.
 9. Themethod of claim 1, wherein said detector obtains said signals from saidplurality of planes (i) as a hologram, (ii) simultaneously from saidplurality of planes, or (ii) sequentially across individual planes ofsaid plurality of planes.
 10. The method of claim 1, wherein said 3Dmatrix comprises a polymeric material.
 11. The method of claim 1,wherein each of said plurality of amplicons is functionalized to formcovalent attachments to said 3D matrix.
 12. The method of claim 1,wherein said signals are obtained at a plurality of planes within said3D matrix simultaneously.
 13. The method of claim 1, wherein saidsignals are obtained while said plurality of amplicons are contactedwith said detectable labels.
 14. The method of claim 1, wherein (d)further comprises using said signals obtained in (c) to identifysequences of said plurality of amplicons, and wherein said 3D volumetricimage of said plurality of amplicons identifies said sequences.
 15. Themethod of claim 1, wherein (c) further comprises (i) contacting saidplurality of amplicons with a plurality of detection probes each havingsubsequences, (ii) sequentially coupling said detectable labels to atleast a subset of said subsequences, and (iii) determining sequences ofsaid subsequences, thereby determining sequences of at least a subset ofsaid plurality of amplicons.
 16. The method of claim 1, wherein saidbiological sample is disposed adjacent to a stage, and wherein duringsaid sequencing, a position of said detector relative to said stage isadjusted to obtain said signals from said plurality of planes.
 17. Themethod of claim 1, wherein said plurality of planes is a plurality offocal planes of said detector.
 18. A system for nucleic acid analysis,comprising: a support configured to retain a biological samplecomprising a plurality of nucleic acid molecules in a three dimensional(3D) matrix, which plurality of nucleic acid molecules have a relative3D spatial relationship; a detector configured to detect one or moresignals from said biological sample; and a computer operatively coupledto said detector, wherein said computer is configured to: (a) subjectsaid plurality of nucleic acid molecules to nucleic acid amplificationunder conditions sufficient to form a plurality of amplicons within said3D matrix, wherein subsequent to formation, said plurality of ampliconsare coupled to said 3D matrix and have said relative 3D spatialrelationship; (b) subject said plurality of amplicons to nucleic acidsequencing while said plurality of amplicons are coupled to said 3Dmatrix, which nucleic acid sequencing comprises (i) sequentiallycontacting said plurality of amplicons with detectable labels and (ii)using said detector to obtain signals corresponding to said detectablelabels from said 3D matrix, which signals are obtained by said detectorfrom a plurality of planes during said nucleic acid sequencing; and (c)use said signals obtained by said detector from said plurality of planesin (b) to generate a 3D volumetric image of said plurality of amplicons,which 3D volumetric image identifies said relative 3D spatialrelationship of said plurality of amplicons.
 19. The system of claim 18,further comprising an optical assembly comprising said detector, whichoptical assembly is configured to adjust a position of said detectorrelative to said stage to obtain said signals from said plurality ofplanes during said nucleic acid sequencing.
 20. The system of claim 19,wherein said optical assembly comprises one or more objective lensesthat are configured to be in optical communication with said support.21. The system of claim 18, further comprising a track and a motorhaving an encoder feedback for adjusting a position of a focal plane ofsaid detector during said nucleic acid sequencing.
 22. The system ofclaim 18, further comprising a mapping unit configured to map a surfaceof said support with respect to one or more motion axes such that saidbiological sample is reproducibly positioned relative to said support.23. The system of claim 22, wherein said mapping unit comprises anautofocus unit that is configured to acquire one or more distancemeasurements between said detector and said sample.
 24. The system ofclaim 22, wherein said mapping unit is configured to compute one or moreoffsets relative to a positional encoder along said one or more motionaxes.
 25. The system of claim 18, further comprising a heating orcooling unit operatively coupled to said support and configured tosubject said plurality of nucleic acid molecules to thermo-cycling tofacilitate said nucleic acid amplification.
 26. The system of claim 18,further comprising a fluidics dispenser configured to deliver programmedvolumes of reagents to said biological sample.
 27. The system of claim18, wherein said plurality of planes is a plurality of focal planes ofsaid detector.
 28. The system of claim 18, wherein a position of saiddetector relative to said stage is adjustable for obtaining said signalsfrom said plurality of planes.
 29. The system of claim 18, wherein saidcomputer is configured to use said signals obtained in (b) to identifysequences of said plurality of amplicons, and wherein said 3D volumetricimage of said plurality of amplicons identifies said sequences.
 30. Thesystem of claim 18, wherein said detector is configured to obtain saidsignals from said plurality of planes (i) as a hologram, (ii)simultaneously from said plurality of planes, or (ii) sequentiallyacross individual planes of said plurality of planes.